Drugs and drug-like molecules can modulate the function of mucosal-associated invariant T cells.

The major-histocompatibility-complex-(MHC)-class-I-related molecule MR1 can present activating and non-activating vitamin-B-based ligands to mucosal-associated invariant T cells (MAIT cells). Whether MR1 binds other ligands is unknown. Here we identified a range of small organic molecules, drugs, drug metabolites and drug-like molecules, including salicylates and diclofenac, as MR1-binding ligands. Some of these ligands inhibited MAIT cells ex vivo and in vivo, while others, including diclofenac metabolites, were agonists. Crystal structures of a T cell antigen receptor (TCR) from a MAIT cell in complex with MR1 bound to the non-stimulatory and stimulatory compounds showed distinct ligand orientations and contacts within MR1, which highlighted the versatility of the MR1 binding pocket. The findings demonstrated that MR1 was able to capture chemically diverse structures, spanning mono- and bicyclic compounds, that either inhibited or activated MAIT cells. This indicated that drugs and drug-like molecules can modulate MAIT cell function in mammals.

A Comprehensive Investigation of Stimulatory Agents on MAIT and Vα7.2+/CD161- T Cell Response and Effects of Immunomodulatory Drugs.

Mucosal-associated invariant T (MAIT) cells, a subset of Vα7.2+ T cells, are a crucial link between innate and adaptive immunity, responding to various stimuli through TCR-dependent and independent pathways. We investigated the responses of MAIT cells and Vα7.2+/CD161- T cells to different stimuli and evaluated the effects of Cyclosporin A (CsA) and Vitamin D3 (VitD). Peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with various agents (PMA/Ionomycin, 5-OP-RU, 5-OP-RU/IL-12/IL-33) with or without CsA and VitD. Flow cytometric analysis assessed surface markers and intracellular cytokine production. Under steady-state conditions, MAIT cells displayed elevated expression of CCR6 and IL-13. They showed upregulated activation and exhaustion markers after activation, producing IFNγ, TNFα, and TNFα/GzB. CsA significantly inhibited MAIT cell activation and cytokine production. Conversely, Vα7.2+/CD161- T cells exhibited distinct responses, showing negligible responses to 5-OP-RU ligand but increased cytokine production upon PMA stimulation. Our study underscores the distinct nature of MAIT cells compared to Vα7.2+/CD161- T cells, which resemble conventional T cells. CsA emerges as a potent immunosuppressive agent, inhibiting proinflammatory cytokine production in MAIT cells. At the same time, VitD supports MAIT cell activation and IL-13 production, shedding light on potential therapeutic avenues for immune modulation.

Chemical Modulators of Mucosal Associated Invariant T Cells.

Over the past decade, we have contributed to the chemistry of microbial natural products and synthetic ligands, related to riboflavin and uracils, that modulate immune cells called mucosal associated invariant T cells (MAIT cells). These highly abundant T lymphocytes were only discovered in 2003 and have become recognized for their importance in mammalian immunology. Unlike other T cells, MAIT cells are not activated by peptide or lipid antigens. In collaboration with immunology and structural biology research groups, we discovered that they are instead activated by unstable nitrogen-containing heterocycles synthesized by bacteria. The most potent naturally occurring activating compound (antigen) is 5-(2-oxopropylideneamino)-d-ribitylaminouracil (5-OP-RU). This compound is an imine (Schiff base) formed through condensation between an intermediate in the biosynthesis of riboflavin (vitamin B2) and a metabolic byproduct of mammalian and microbial glycolysis. Although it is very unstable in water due to intramolecular ring closure or hydrolysis, we were able to develop a non-enzymatic synthesis that yields a pure kinetically stable compound in a nonaqueous solvent. This compound has revolutionized the study of MAIT cell immunology due to its potent activation (EC50 = 2 pM) of MAIT cells and its development into immunological reagents for detecting and characterizing MAIT cells in tissues. MAIT cells are now linked to key physiological processes and disease, including antibacterial defense, tissue repair, regulation of graft-vs-host disease, gastritis, inflammatory bowel diseases, and cancer. 5-OP-RU activates MAIT cells and, like a vaccine, has been shown to protect mice from bacterial infections and cancers. Mechanistic studies on the binding of 5-OP-RU to its dual protein targets, the major histocompatibility complex class I related protein (MR1) and the MAIT cell receptor (MAIT TCR), have involved synthetic chemistry, 2D 1H NMR spectroscopy, mass spectrometry, computer modeling and molecular dynamics simulations, biochemical, cellular, and immunological assays, and protein structural biology. These combined studies have revealed structural influences for 5-OP-RU in solution on protein binding and antigen presentation and potency; informed the development of potent (EC50 = 2 nM) and water stable analogues; led to fluorescent analogues for detecting and tracking binding proteins in and on cells; and enabled discovery of drugs and drug-like molecules that bind MR1 and modulate MAIT cell function. MAIT cells offer new opportunities for chemical synthesis to enhance the stability, potency, selectivity, and bioavailability of small molecule ligands for MR1 or MAIT TCR proteins, and to contribute to the understanding of T cell immunity and the development of prospective new immunomodulating medicines.

Alcohol-associated intestinal dysbiosis alters mucosal-associated invariant T-cell phenotype and function.

BACKGROUND: Chronic alcohol consumption is associated with a compromised innate and adaptive immune responses to infectious disease. Mucosa-associated invariant T (MAIT) cells play a critical role in antibacterial host defense. However, whether alcohol-associated deficits in innate and adaptive immune responses are mediated by alterations in MAIT cells remains unclear. METHODS: To investigate the impact of alcohol on MAIT cells, mice were treated with binge-on-chronic alcohol for 10 days and sacrificed at day 11. MAIT cells in the barrier organs (lung, liver, and intestine) were characterized by flow cytometry. Two additional sets of animals were used to examine the involvement of gut microbiota on alcohol-induced MAIT cell changes: (1) Cecal microbiota from alcohol-fed (AF) mice were adoptive transferred into antibiotic-pretreated mice and (2) AF mice were treated with antibiotics during the experiment. MAIT cells in the barrier organs were measured via flow cytometry. RESULTS: Binge-on-chronic alcohol feeding led to a significant reduction in the abundance of MAIT cells in the barrier tissues. However, CD69 expression on tissue-associated MAIT cells was increased in AF mice compared with pair-fed (PF) mice. The expression of Th1 cytokines and the corresponding transcriptional factor was tissue specific, showing downregulation in the intestine and increases in the lung and liver in AF animals. Transplantation of fecal microbiota from AF mice resulted in a MAIT cell profile aligned to that of AF mouse donor. Antibiotic treatment abolished the MAIT cell differences between AF and PF animals. CONCLUSION: MAIT cells in the intestine, liver, and lung are perturbed by alcohol use and these changes are partially attributable to alcohol-associated dysbiosis. MAIT cell dysfunction may contribute to alcohol-induced innate and adaptive immunity and consequently end-organ pathophysiology.

IL-33 Enhances IFNγ and TNFα Production by Human MAIT Cells: A New Pro-Th1 Effect of IL-33.

Mucosal-associated invariant T (MAIT) cells represent a distinct T cell population restricted by the MHC-class-I-related molecule, MR1, which recognizes microbial-derived vitamin B2 (riboflavin) metabolites. Their abundance in humans, together with their ability to promptly produce distinct cytokines including interferon γ (IFNγ) and tumor necrosis factor α (TNFα), are consistent with regulatory functions in innate as well as adaptive immunity. Here, we tested whether the alarmin interleukin 33 (IL-33), which is secreted following inflammation or cell damage, could activate human MAIT cells. We found that MAIT cells stimulated with IL-33 produced high levels of IFNγ, TNFα and Granzyme B (GrzB). The action of IL-33 required IL-12 but was independent of T cell receptor (TCR) cross-linking. MAIT cells expressed the IL-33 receptor ST2 (suppression of tumorigenicity 2) and upregulated Tbet (T-box expressed in T cells) in response to IL-12 or IL-33. Electronically sorted MAIT cells also upregulated the expression of CCL3 (Chemokine C-C motif ligand 3), CD40L (CD40 Ligand), CSF-1 (Colony Stimulating Factor 1), LTA (Lymphotoxin-alpha) and IL-2RA (IL-2 receptor alpha chain) mRNAs in response to IL-33 plus IL-12. In conclusion, IL-33 combined with IL-12 can directly target MAIT cells to induce their activation and cytokine production. This novel mechanism of IL-33 activation provides insight into the mode of action by which human MAIT cells can promote inflammatory responses in a TCR-independent manner.

Efficient 5-OP-RU-Induced Enrichment of Mucosa-Associated Invariant T Cells in the Murine Lung Does Not Enhance Control of Aerosol Mycobacterium tuberculosis Infection.

Mucosa-associated invariant T (MAIT) cells are an innate-like T cell subset in mammals that recognize microbial vitamin B metabolites presented by the evolutionarily conserved major histocompatibility complex class I (MHC I)-related molecule, MR1. Emerging data suggest that MAIT cells may be an attractive target for vaccine-induced protection against bacterial infections because of their rapid cytotoxic responses at mucosal services to a widely conserved bacterial ligand. In this study, we tested whether a MAIT cell priming strategy could protect against aerosol Mycobacterium tuberculosis infection in mice. Intranasal costimulation with the lipopeptide Toll-like receptor (TLR)2/6 agonist, Pam2Cys (P2C), and the synthetic MR1 ligand, 5-OP-RU, resulted in robust expansion of MAIT cells in the lung. Although MAIT cell priming significantly enhanced MAIT cell activation and expansion early after M. tuberculosis challenge, these MAIT cells did not restrict M. tuberculosis bacterial load. MAIT cells were depleted by the onset of the adaptive immune response, with decreased detection of granzyme B+ and gamma interferon (IFN-γ)+ MAIT cells relative to that in uninfected P2C/5-OP-RU-treated mice. Decreasing the infectious inoculum, varying the time between priming and aerosol infection, and testing MAIT cell priming in nitric oxide synthase 2 (NOS2)-deficient mice all failed to reveal an effect of P2C/5-OP-RU-induced MAIT cells on M. tuberculosis control. We conclude that intranasal MAIT cell priming in mice induces early MAIT cell activation and expansion after M. tuberculosis exposure, without attenuating M. tuberculosis growth, suggesting that MAIT cell enrichment in the lung is not sufficient to control M. tuberculosis infection.

MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression.

Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a "cytokine storm" with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm's initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host's ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vß-specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses.

Mucosal-Associated Invariant T Cells in Tumors of Epithelial Origin.

Mucosal-associated invariant T (MAIT) cells are innate T lymphocytes that circulate in blood and also reside in mucosal tissues. Blood MAIT cells are typically highly Th1-polarized, while those in mucosal tissues include both Th1- and Th17-polarized subsets. MAIT cells mount cytokine and cytolytic responses as a result of T cell receptor (TCR)-mediated recognition of microbially derived metabolites of riboflavin (vitamin B2) presented by the MR1 antigen-presenting molecule. Additionally, MAIT cells can be activated by inflammatory cytokines produced by antigen-presenting cells (APCs) that have been exposed to pathogen-associated molecular patterns (PAMPs). Since the antigenic metabolites of riboflavin recognized by MAIT cells are produced by many microorganisms, including pathogens as well as non-pathogenic colonists, the inflammatory state of the tissue may be a key feature that determines the nature of MAIT cell responses. Under normal conditions where inflammatory cytokines are not produced, MAIT cell responses to microbial metabolites may simply serve to help maintain a healthy balance between epithelial cells and microbial colonists. In contrast, in situations where inflammatory cytokines are produced (e.g., pathogenic infection or damage to epithelial tissue), MAIT cell responses may be more potently pro-inflammatory. Since chronic inflammation and microbial drivers are associated with tumorigenesis and also trigger MAIT cell responses, the nexus of MAIT cells, local microbiomes, and epithelial cells may play an important role in epithelial carcinogenesis. This chapter reviews current information about MAIT cells and epithelial tumors, where the balance of evidence suggests that enrichment of Th17-polarized MAIT cells at tumor sites associates with poor patient prognosis. Studying the role of MAIT cells and their interactions with resident microbes offers a novel view of the biology of epithelial tumor progression and may ultimately lead to new approaches to target MAIT cells clinically.

The effect of MR1 ligand glyco-analogues on mucosal-associated invariant T (MAIT) cell activation.

Mucosal-associated invariant T (MAIT) cells are a subset of recently identified innate-like T lymphocytes that appear to play an important role in many pathologies ranging from viral and bacterial infection, to autoimmune disorders and cancer. MAIT cells are activated via the presentation of ligands by MR1 on antigen presenting cells to the MAIT T cell receptor (TCR), however few studies have explored the effects of systematic changes to the ligand structure on MR1 binding and MAIT cell activation. Herein, we report on the first study into the effects of changes to the sugar motif in the known MAIT cell agonists 7-hydroxy-6-methyl-8-d-ribityllumazine (RL-6-Me-7-OH) and 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU). Tetramer staining of MAIT cells revealed that the absence of the 2'-hydroxy group on the sugar backbone of lumazines improved MR1-MAIT TCR binding, which could be rationalised using computational docking studies. Although none of the lumazines activated MAIT cells, all 5-OP-RU analogues showed significant MAIT cell activation, with several analogues exhibiting comparable activity to 5-OP-RU. Docking studies with the 5-OP-RU analogues revealed different interactions between the sugar backbone and MR1 and the MAIT TCR compared to those observed for the lumazines and confirmed the importance of the 2'-hydroxy group for ligand binding and activity. Taken together, this information will assist in the development of future potent agonists and antagonists of MAIT cells.

Intra-Hepatic Depletion of Mucosal-Associated Invariant T Cells in Hepatitis C Virus-Induced Liver Inflammation.

BACKGROUND & AIMS: Chronic hepatitis affects phenotypes of innate and adaptive immune cells. Mucosal-associated invariant T (MAIT) cells are enriched in the liver as compared with the blood, respond to intra-hepatic cytokines, and (via the semi-invariant T-cell receptor) to bacteria translocated from the gut. Little is known about the role of MAIT cells in livers of patients with chronic hepatitis C virus (HCV) infection and their fate after antiviral therapy. METHODS: We collected blood samples from 42 patients with chronic HCV infection who achieved a sustained virologic response after 12 weeks of treatment with sofosbuvir and velpatasvir. Mononuclear cells were isolated from blood before treatment, at weeks 4 and 12 during treatment, and 24 weeks after the end of treatment. Liver biopsies were collected from 37 of the patients prior to and at week 4 of treatment. Mononuclear cells from 56 blood donors and 10 livers that were not suitable for transplantation were used as controls. Liver samples were assessed histologically for inflammation and fibrosis. Mononuclear cells from liver and blood were studied by flow cytometry and analyzed for responses to cytokine and bacterial stimulation. RESULTS: The frequency of MAIT cells among T cells was significantly lower in blood and liver samples of patients with HCV infection than of controls (median, 1.31% vs 2.32% for blood samples, P = .0048; and median, 4.34% vs 13.40% for liver samples, P = .001). There was an inverse correlation between the frequency of MAIT cells in the liver and histologically determined levels of liver inflammation (r = -.5437, P = .0006) and fibrosis (r = -.5829, P = .0002). MAIT cells from the liver had higher levels of activation and cytotoxicity than MAIT cells from blood (P < .0001). Production of interferon gamma by MAIT cells was dependent on monocyte-derived interleukin 18, and was reduced in patients with HCV infection in response to T-cell receptor-mediated but not cytokine-mediated stimulation, as compared with controls. Anti-viral therapy rapidly decreased liver inflammation and MAIT cell activation and cytotoxicity, and increased the MAIT cell frequency among intra-hepatic but not blood T cells. The MAIT cell response to T-cell receptor-mediated stimulation did not change during the 12 weeks of antiviral therapy. CONCLUSIONS: In analyses of paired blood and liver samples from patients with chronic HCV infection before, during, and after antiviral therapy with sofosbuvir and velpatasvir, we found that intrahepatic MAIT cells are activated by monocyte-derived cytokines and depleted in HCV-induced liver inflammation.

Steroid-induced Deficiency of Mucosal-associated Invariant T Cells in the Chronic Obstructive Pulmonary Disease Lung. Implications for Nontypeable Haemophilus influenzae Infection.

RATIONALE: Mucosal-associated invariant T (MAIT) cells are a recently described abundant, proinflammatory T-cell subset with unknown roles in pulmonary immunity. Nontypeable Haemophilus influenzae (NTHi) is the leading bacterial pathogen during chronic obstructive pulmonary disease (COPD) exacerbations and is a plausible target for MAIT cells. OBJECTIVES: To investigate whether MAIT cells respond to NTHi and the effects of inhaled corticosteroids (ICS) on their frequency and function in COPD. METHODS: Eleven subjects with COPD receiving ICS, 8 steroid-naive subjects with COPD, and 21 healthy control subjects underwent phlebotomy, sputum induction, bronchoalveolar lavage, and endobronchial biopsy. Pulmonary and monocyte-derived macrophages were cultured in vitro with NTHi. MEASUREMENTS AND MAIN RESULTS: Frequencies of Vα7.2+CD161+ MAIT cells, surface expression of the major histocompatibility complex-related protein 1 (MR1), and intracellular IFN-γ expression were measured by flow cytometry. MAIT-cell frequencies were reduced in peripheral blood of ICS-treated subjects with COPD (median 0.38%; interquartile range [IQR], 0.25-0.96) compared with healthy control subjects (1.8%; IQR, 1.4-2.5; P = 0.001) or steroid-naive patients with COPD (1.8%; IQR, 1.2-2.3; P = 0.04). MAIT cells were reduced in bronchial biopsies from subjects with COPD treated with steroids (0.73%; IQR, 0.46-1.3) compared with healthy control subjects (4.0%; IQR, 1.6-5.0; P = 0.02). Coculture of live NTHi increased macrophage surface expression of MR1 and induced IFN-γ from CD4 cells and CD8 cells, but most potently from MAIT cells (median IFN-γ-positive frequencies, 2.9, 8.6, and 27.6%, respectively). In vitro fluticasone and budesonide reduced MR1 surface expression twofold and decreased NTHi-induced IFN-γ secretion eightfold. CONCLUSIONS: MAIT cells are deficient in blood and bronchial tissue in steroid-treated, but not steroid-naive, COPD. NTHi constitutes a target for pulmonary MAIT-cell immune responses, which are significantly impaired by corticosteroids.

Chemical insights into the search for MAIT cells activators.

Mucosal-associated invariant T cells (MAIT cells) represent a potential therapeutic target as they can tune or enhance immune responses. They recognise and become activated by antigens, presented by the monomorphic MHC-I related molecule, MR1. To assess the significance of MAIT cells in human diseases, a better understanding of the MAIT cell-MR1-antigen interaction is imperative. Easy access to MR1 ligands and MAIT cells activators can help achieve this. In this review, we summarise current literature that has identified the natural ligands and drug-like molecules that activate MAIT cells and provide insight into their key molecular interactions with MR1 and MAIT T cell receptors (TCRs). We focus on the progress made in synthesizing and isolating 5-amino-6-d-ribitylaminouracil (5-A-RU), a key precursor in the synthesis of the known natural ligands, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil(5-OP-RU) and 5-(2-oxoethylideneamino)-6-d-ribitylaminouracil (5-OE-RU), and also on the stabilisation and optimisation of the latter compounds.

Oral Selective TLR8 Agonist Selgantolimod Induces Multiple Immune Cell Responses in Humans.

TLR8 agonists have the potential for use as immunomodulatory components in therapeutic modalities for viral infections such as chronic HBV (CHB) and HIV. In this study, using peripheral blood samples from a phase 1a clinical trial, we examined the acute effects of a single oral administration of a selective TLR8 agonist on immune cell phenotypes. Administration of the TLR8 agonist selgantolimod (SLGN) in healthy individuals resulted in alteration in frequencies of peripheral blood monocytes, pDCs, mDCs and MAIT cells. Frequencies of mDCs and lymphoid cells significantly reduced after 8 h of SLGN administration, whereas pDC frequencies significantly increased, with changes possibly reflecting migration of different cell types between peripheral and tissue compartments in response to the agonist. Myeloid cell activation was evident by an upregulated expression of co-stimulatory molecules CD40 and CD86 accompanied by the production of IL-6 and IL-18 from these cells. Concomitantly, there was induction of the early activation marker CD69 on innate and adaptive lymphoid cells, including MAIT and NK cell subsets. Further, these activated lymphoid cells had enhanced expression of the effector molecules granzyme B and perforin. Microarray analysis of isolated lymphocytes and monocytes from baseline and post-SLGN treatment revealed changes in expression of genes involved in cellular response to cytokine stimulus, innate immune response, myeloid cell differentiation and antigen receptor-mediated signaling pathway. In a preliminary analysis of samples from CHB patients treated with selgantolimod, activation of innate and adaptive lymphocytes was evident. In conclusion, this first in-human study shows that selgantolimod administration in humans results in activation of multiple immune cell responses with antiviral potential.

Functional and Activation Profiles of Mucosal-Associated Invariant T Cells in Patients With Tuberculosis and HIV in a High Endemic Setting.

Background: MAIT cells are non-classically restricted T lymphocytes that recognize and rapidly respond to microbial metabolites or cytokines and have the capacity to kill bacteria-infected cells. Circulating MAIT cell numbers generally decrease in patients with active TB and HIV infection, but findings regarding functional changes differ. Methods: We conducted a cross-sectional study on the effect of HIV, TB, and HIV-associated TB (HIV-TB) on MAIT cell frequencies, activation and functional profile in a high TB endemic setting in South Africa. Blood was collected from (i) healthy controls (HC, n = 26), 24 of whom had LTBI, (ii) individuals with active TB (aTB, n = 36), (iii) individuals with HIV infection (HIV, n = 50), 37 of whom had LTBI, and (iv) individuals with HIV-associated TB (HIV-TB, n = 26). All TB participants were newly diagnosed and sampled before treatment, additional samples were also collected from 18 participants in the aTB group after 10 weeks of TB treatment. Peripheral blood mononuclear cells (PBMC) stimulated with BCG-expressing GFP (BCG-GFP) and heat-killed (HK) Mycobacterium tuberculosis (M.tb) were analyzed using flow cytometry. MAIT cells were defined as CD3+ CD161+ Vα7.2+ T cells. Results: Circulating MAIT cell frequencies were depleted in individuals with HIV infection (p = 0.009). MAIT cells showed reduced CD107a expression in aTB (p = 0.006), and reduced IFNγ expression in aTB (p < 0.001) and in HIV-TB (p < 0.001) in response to BCG-GFP stimulation. This functional impairment was coupled with a significant increase in activation (defined by HLA-DR expression) in resting MAIT cells from HIV (p < 0.001), aTB (p = 0.019), and HIV-TB (p = 0.005) patients, and higher HLA-DR expression in MAIT cells expressing IFNγ in aTB (p = 0.009) and HIV-TB (p = 0.002) after stimulation with BCG-GFP and HK-M.tb. After 10 weeks of TB treatment, there was reversion in the observed functional impairment in total MAIT cells, with increases in CD107a (p = 0.020) and IFNγ (p = 0.010) expression. Conclusions: Frequencies and functional profile of MAIT cells in response to mycobacterial stimulation are significantly decreased in HIV infected persons, active TB and HIV-associated TB, with a concomitant increase in MAIT cell activation. These alterations may reduce the capacity of MAIT cells to play a protective role in the immune response to these two pathogens.

Diverse Mucosal-Associated Invariant TCR Usage in HIV Infection.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that specifically target bacterial metabolites but are also identified as innate-like sensors of viral infection. Individuals with chronic HIV-1 infection have lower numbers of circulating MAIT cells compared with healthy individuals, yet the features of the MAIT TCR repertoire are not well known. We isolated and stimulated human PBMCs from healthy non-HIV-infected donors (HD), HIV-infected progressors on antiretroviral therapy, and HIV-infected elite controllers (EC). We sorted MAIT cells using flow cytometry and used a high-throughput sequencing method with bar coding to link the expression of TCRα, TCRß, and functional genes of interest at the single-cell level. We show differential patterns of MAIT TCR usage among the groups. We observed expansions of certain dominant MAIT clones in HIV-infected individuals upon Escherichia coli stimulation, which was not observed in clones of HD. We also found different patterns of CDR3 amino acid distributions among the three groups. Furthermore, we found blunted expression of phenotypic genes in HIV individuals; most notably, HD mounted a robust IFNG response to stimulation, whereas both HIV-infected progressors and EC did not. In conclusion, our study describes the diverse MAIT TCR repertoire of persons with chronic HIV-1 infection and suggest that MAIT clones of HIV-infected persons may be primed for expansion more than that of noninfected persons. Further studies are needed to examine the functional significance of unique MAIT cell TCR usage in EC.

Functional inactivation of pulmonary MAIT cells following 5-OP-RU treatment of non-human primates.

Targeting MAIT cells holds promise for the treatment of different diseases and infections. We previously showed that treatment of Mycobacterium tuberculosis infected mice with 5-OP-RU, a major antigen for MAIT cells, expands MAIT cells and enhances bacterial control. Here we treated M. tuberculosis infected rhesus macaques with 5-OP-RU intratracheally but found no clinical or microbiological benefit. In fact, after 5-OP-RU treatment MAIT cells did not expand, but rather upregulated PD-1 and lost the ability to produce multiple cytokines, a phenotype resembling T cell exhaustion. Furthermore, we show that vaccination of uninfected macaques with 5-OP-RU+CpG instillation into the lungs also drives MAIT cell dysfunction, and PD-1 blockade during vaccination partly prevents the loss of MAIT cell function without facilitating their expansion. Thus, in rhesus macaques MAIT cells are prone to the loss of effector functions rather than expansion after TCR stimulation in vivo, representing a significant barrier to therapeutically targeting these cells.

Activating Mucosal-Associated Invariant T Cells Induces a Broad Antitumor Response.

Mucosal-associated invariant T (MAIT) cells are MR1-restricted innate-like T cells that recognize non-peptide antigens including riboflavin derivates. Although in vitro-activated MAIT cells show antitumor activity, the in vivo role of MAIT cells in cancer is still unclear. Here, we have shown that MAIT cells have antitumor function in vivo when activated by a combination of the synthetic riboflavin synthesis pathway-derived antigen 5-OP-RU [5-(2-oxopropylideneamino)-6-D-ribitylaminouracil] and the Toll-like receptor 9 (TLR9) agonist CpG. Coadministration of 5-OP-RU and CpG induced strong systemic in vivo expansion and activation of MAIT cells with high CD69 expression, pronounced effector memory phenotype, and upregulated levels of effector molecules including IFNγ, granzyme B, and perforin. Activated and expanded MAITs induced a potent and broad antitumor immune response in murine models of liver metastasis and hepatocellular carcinoma, lung metastasis, and subcutaneous tumors in two different mouse strains. Such tumor inhibition was absent in MAIT-deficient Mr1 -/- mice. CRISPR/Cas9-mediated MR1 knockout in tumor cells did not affect efficacy of this MAIT-directed immunotherapy, pointing toward an indirect mechanism of action. Our findings suggest that MAIT cells are an attractive target for cancer immunotherapy.See related Spotlight by Lantz, p. 996.

Chronic stress physically spares but functionally impairs innate-like invariant T cells.

The deleterious effects of psychological stress on mainstream T lymphocytes are well documented. However, how stress impacts innate-like T cells is unclear. We report that long-term stress surprisingly abrogates both T helper 1 (TH1)- and TH2-type responses orchestrated by invariant natural killer T (iNKT) cells. This is not due to iNKT cell death because these cells are unusually refractory to stress-inflicted apoptosis. Activated iNKT cells in stressed mice exhibit a "split" inflammatory signature and trigger sudden serum interleukin-10 (IL-10), IL-23, and IL-27 spikes. iNKT cell dysregulation is mediated by cell-autonomous glucocorticoid receptor signaling and corrected upon habituation to predictable stressors. Importantly, under stress, iNKT cells fail to potentiate cytotoxicity against lymphoma or to reduce the burden of metastatic melanoma. Finally, stress physically spares mouse mucosa-associated invariant T (MAIT) cells but hinders their TH1-/TH2-type responses. The above findings are corroborated in human peripheral blood and hepatic iNKT/MAIT cell cultures. Our work uncovers a mechanism of stress-induced immunosuppression.

Agonistic or antagonistic mucosal-associated invariant T (MAIT) cell activity is determined by the 6-alkylamino substituent on uracil MR1 ligands.

Mucosal-associated invariant T (MAIT) are a subset of innate-like T cells that are activated by uracil ligands presented by MR1. For the first time, we demonstrate that changes to the 6-aminoalkyl chain on uracil agonist 5-OP-RU can determine agonistic or antagonistic MAIT cell activity. Insomuch, a simplified agonist with a functional profile similar to 5-OP-RU, and a new structural class of antagonist that exhibits similar activity to known MAIT cell antagonist Ac-6-FP, were identified.